Calcineurin Negatively Regulates TLR-Mediated Activation Pathways Young Jun Kang, Brenda Kusler, Motoyuki Otsuka, Michael Hughes, Nobutaka Suzuki, Shinobu Suzuki, Wen-Chen Yeh, This information is current as Shizuo Akira, Jiahuai Han and Patricia P. Jones of September 26, 2021. J Immunol 2007; 179:4598-4607; ; doi: 10.4049/jimmunol.179.7.4598 http://www.jimmunol.org/content/179/7/4598 Downloaded from References This article cites 61 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/179/7/4598.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Calcineurin Negatively Regulates TLR-Mediated Activation Pathways1 Young Jun Kang,2,3* Brenda Kusler,* Motoyuki Otsuka,† Michael Hughes,* Nobutaka Suzuki,‡ Shinobu Suzuki,‡ Wen-Chen Yeh,‡ Shizuo Akira,§ Jiahuai Han,† and Patricia P. Jones3* In innate immunity, microbial components stimulate macrophages to produce antimicrobial substances, cytokines, other proin- flammatory mediators, and IFNs via TLRs, which trigger signaling pathways activating NF-␬B, MAPKs, and IFN response factors. We show in this study that, in contrast to its activating role in T cells, in macrophages the protein phosphatase calcineurin negatively regulates NF-␬B, MAPKs, and IFN response factor activation by inhibiting the TLR-mediated signaling pathways. Evidence for this novel role for calcineurin was provided by the findings that these signaling pathways are activated when calcineurin is inhibited either by the inhibitors cyclosporin A or FK506 or by small interfering RNA-targeting calcineurin, and that activation of these pathways by TLR ligands is inhibited by the overexpression of a constitutively active form of calcineurin. Downloaded from We further found that I␬B-␣ degradation, MAPK activation, and TNF-␣ production by FK506 were reduced in macrophages from mice deficient in MyD88, Toll/IL-1R domain-containing adaptor-inducing IFN-␤ (TRIF), TLR2, or TLR4, whereas macrophages from TLR3-deficient or TLR9 mutant mice showed the same responses to FK506 as those of wild-type cells. Biochemical studies indicate that calcineurin interacts with MyD88, TRIF, TLR2, and TLR4, but not with TLR3 or TLR9. Collectively, these results suggest that calcineurin negatively regulates TLR-mediated activation pathways in macrophages by inhibiting the adaptor proteins MyD88 and TRIF, and a subset of TLRs. The Journal of Immunology, 2007, 179: 4598–4607. http://www.jimmunol.org/ n innate immunity, microbial components such as LPS, li- TLR10 has been identified in humans, but its ligand specificity is poprotein, peptidoglycan, unmethylated CpG DNA motifs, unknown (3, 12). TLR11, which is only expressed in mice, but not I flagellin, and viral dsRNA stimulate macrophages to produce in humans, is reported to be important in the immune response proinflammatory cytokines such as TNF-␣, IL-1, IL-6, and IL-12, against the uropathogenic bacteria (13). TLRs are expressed extra- as well as chemokines, inducible NO synthase, other antimicrobial or intracellularly. Although some TLRs (TLRs 1, 2, 4, 5, and 6) are responses, and coreceptor molecules. The interactions of the mi- expressed on the cell surface, others (TLRs 3, 7, 8, and 9) are crobial components with TLRs lead to the activation of the tran- found in intracellular compartments such as endosomes (14). Al- by guest on September 26, 2021 scription factor NF-␬B (1–3), which is required for the induction though the receptor for each ligand is specific, the downstream of these effector functions. The TLR-activated signaling pathway events are mediated by two shared signaling pathways. The first, is evolutionarily conserved, because activation of antimicrobial which is MyD88 dependent, is activated by all TLRs, except gene expression in insects by bacteria or fungi occurs via a ho- TLR3, and also by IL-1R (15, 16). Activated receptors induce the mologous pathway triggered by Toll receptors that leads to the formation of a complex of the adaptor protein MyD88 (17), which activation of the dorsal/rel family of NF-␬B homologues (4). recruits IL-1R-associated kinase (IRAK)4-4, which in turn binds Bacterial components activate specific TLRs on the plasma and activates IRAK-1, which then undergoes autophosphorylation membrane, as follows: lipid A component of bacterial endotoxin (18, 19). Following activation, phosphorylated IRAK-1 binds LPS activates TLR4 (5); lipoprotein and peptidoglycan activate TNFR-associated factor (TRAF)6 (20, 21), which in turn forms a TLR2 (6); bacterial CpG DNA activates TLR9 (7); flagellin acti- complex at the plasma membrane with TGF-␤-activated kinase vates TLR5 (8); viral dsRNA activates TLR3 (9); and ssRNA and (TAK)1, TAK1-binding protein (TAB)1, and TAB2, which in- synthetic nucleotide derivatives activate TLR7 and 8 (10, 11). duces the phosphorylation of TAB2 and TAK1. Activated TAK1 phosphorylates and activates the I␬B kinases (IKKs). The NF-␬B p50:p65 complex is normally sequestered in an inactive form in *Department of Biological Sciences, Stanford University, Stanford, CA 94305; †De- ␬ partment of Immunology, The Scripps Research Institute, La Jolla, CA 92037; ‡Ad- the cytoplasm through interaction with I Bs. Activated IKK phos- vanced Medical Discovery Institute, University Health Network and Department of phorylates I␬Bs, which are then ubiquitinated and degraded by Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; and §Depart- proteasomes (22–24). The free NF-␬B complex translocates to the ment of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan nucleus and activates the transcription of target genes. TAK1 also Received for publication March 29, 2007. Accepted for publication July 11, 2007. activates pathways leading to the activation of ERK, JNK, and p38 MAPK pathways (25), which activate downstream transcription The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance factors that contribute to inflammatory gene expression. with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by funds from Stanford University (to P.P.J.). 2 Current address: Department of Immunology, The Scripps Research Institute, 10550 4 Abbreviations used in this paper: IRAK, IL-1R-associated kinase; AKAP, A-kinase North Torrey Pines Road, La Jolla, CA 92037. anchoring protein; CsA, cyclosporin A; IKK, I␬B kinase; IP-10, IFN-␥-inducible 3 Address correspondence and reprint requests to Dr. Young Jun Kang, 10550 North protein-10; IRF, IFN response factor; siRNA, small interfering RNA; TAB, TGF-␤- Torrey Pines Road, IMM-32, Department of Immunology, The Scripps Research activated kinase 1-binding protein; TAK, TGF-␤-activated kinase; TRAF, TNFR- Institute, La Jolla, CA 92037; E-mail address: [email protected] or Dr. Patricia P. associated factor; TRIF, Toll/IL-1R domain-containing adaptor-inducing IFN-␤. Jones, 371 Serra Mall, Gilbert Building, Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020; E-mail address: [email protected] Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 4599 The second pathway, which is MyD88 independent, is triggered University, Stanford, CA). FLAG-tagged human TRIF was cloned into by TLR4 (which also activates the MyD88-dependent pathway) pcDNA expression vector (Invitrogen Life Technologies). The pEGFP-C1 vector was purchased from BD Clontech; pNF-␬B-luciferase vector was and TLR3, leading to the activation of the transcription factor IFN ␤ ␤ purchased from Stratagene; pSV- -galactosidase vector was purchased response factor (IRF)3 and IFN- production (9, 26, 27), which from Promega. Control and CnA (the catalytic subunit of calcineurin, ␣ contributes to innate antiviral responses. The Toll/IL-1R domain- isoform)-targeting siRNA were purchased from Santa Cruz Biotechnology. containing adaptor-inducing IFN-␤ (TRIF) was identified as an Calcineurin inhibitor FK506 was provided by Fujisawa Pharmaceutical adaptor for TLR3 and TLR4 for the MyD88-independent pathway or purchased from LC Laboratories. LPS (Escherichia coli O111:B4) was obtained from Sigma-Aldrich and List Biological Laboratories; soluble (28, 29). peptidoglycan (Staphylococcus aureus) was purchased from Fluka; cal- Calcineurin is a serine/threonine phosphatase that consists of a cineurin inhibitor CsA and poly(I:C) were purchased from Sigma-Aldrich. catalytic subunit, CnA, and a regulatory subunit, CnB. It partici- Mouse rTNF-␣ was purchased from Roche Molecular Biochemicals; hu- pates